Cell cycle progression is controlled by cyclin-dependent kinases (Cdks), the activity of which is positively regulated by cyclins and negatively regulated by Cdk-inhibitors. In late G1 phase of the cell cycle, cyclin E achieves maximal expression level (Koff, et al. (1992) Science 257(5077):1689-94). Cyclin E binds to and activates cyclin-dependent kinase 2 (Cdk2) to promote G1/S transition (Koff, et al. (1992) supra). Cyclin E abundance and cyclin E-Cdk activity are each diminished at the G2/M phase. The amount and timing of cyclin E-Cdk2 activity are both precisely regulated to assure the normal progression of the cell cycle. Constitutive cyclin E overexpression shortens the G1 phase, impairs S-phase progression, and causes chromosomal instability (Ohtsubo, et al. (1995) Mol. Cell Biol. 15:2612-2624; Spruck, et al. (1999) Nature 401:297-300). In contrast, inhibition of cyclin E in G1 phase of the cell cycle blocks the occurrence of S-phase (Ohtsubo, et al. (1995) supra).
Cyclin E is regulated primarily at the levels of gene transcription and by ubiquitin-dependent proteolysis. Cyclin E is a transcriptional target of E2F family members, the function of which is repressed by association with the retinoblastoma (Rb) gene product. Phosphorylation of Rb by D-type cyclin-associated Cdk4/6 can release E2F thereby activating cyclin E expression (Coqueret (2002) Gene 299:35-55). Cul3 promotes ubiquitination of free cyclin E that is unbound to Cdk2 (Clurman, et al. (1996) Genes Dev. 10:1979-90; Singer, et al. (1999) Genes Dev. 13:2375-87). Ubiquitination of Cdk2 bound cyclin E is dependent on phosphorylation of threonine 380 (Won and Reed (1996) EMBO J. 15:4182-93) and threonine 62 (Strohmaier, et al. (2001) Nature 413(6853):316-22). Phosphorylation of these amino acids allows cyclin E to be recognized by Fbw7 (hCdc4) (Strohmaier, et al. (2001) supra), which is a phosphoepitope-specific substrate recognition component of SCF ubiquitin ligases.
During lung carcinogenesis, accumulation of genetic and epigenetic alterations causes invasive or metastatic lung cancers (Dragnev, et al. (2003) Cancer Biol. Ther. 2:S150-6). Lung cancer is the leading cause of cancer mortality for men and women in United States. Key molecular changes, especially at early stages of carcinogenesis, represent potential pharmacological targets for lung cancer chemoprevention and therapy. Aberrant expression of cyclin E has been frequently observed in premalignant lung lesions (Lonardo, et al. (1999) Cancer Res. 59:2470-6), indicating that this is an early step in lung carcinogenesis. Overexpression of cyclin E also occurs in overt non-small cell lung cancers (NSCLCs) and has prognostic significance in NSCLCs (Fukuse, et al. (2000) Cancer Res. 60:242-4; Muller-Tidow, et al. (2001) Cancer Res. 61:647-53; Dosaka-Akita, et al. (2001) Cancer Res. 61:2500-4). The mechanisms responsible for cyclin E deregulation in lung cancer are unknown. However, several mechanisms have been proposed in other tumor cells including gene amplification (Cassia, et al. (2003) J. Pathol. 201:589-95; Schraml, et al. (2003) J. Pathol. 200:375-82), mutation in the F-box protein hCDC4 (Strohmaier, et al. (2001) supra; Spruck, et al. (2002) Cancer Res. 62:4535-9; Rajagopalan, et al. (2004) Nature 428:77-81), as well as proteolytic processing of full-length cyclin E at two sites in the amino-terminus by an elastase-like protease (Harwell, et al. (2000) Cancer Res. 60:481-9; Porter, et al. (2001) Mol. Cell Biol. 21:6254-69).
U.S. patent application Ser. No. 10/392,113 teaches a cell-based assay system comprising at least two manipulated (e.g., upregulated or down regulated) genes such as Cyclin E, Cyclin D, and the like, wherein the genes are involved in a disease state such as cancer for use in identifying a therapeutic reagent.
Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention (Freemantle, et al. (2003) Oncogene 22:7305-15). The retinoid role in cancer chemoprevention has been highlighted by in vitro studies, preclinical animal model experiments, epidemiological evidence, and results of clinical trials wherein certain premalignant or second malignancies have been treated (Freemantle, et al. (2003) supra). All-trans-retinoic acid (RA) has been used as a pharmacologic tool to investigate RA chemopreventive mechanisms. A chemopreventive cell model has been established wherein immortalized BEAS-2B human bronchial epithelial cells are exposed to the carcinogen N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Langenfeld, et al. (1996) Oncogene 13:1983-90). NNK-treated cells undergo cellular transformation resulting in increased proliferation, anchorage-independent growth, increased cyclin E expression and tumor formation in athymic mice (Langenfeld, et al. (1996) supra). RA antagonizes NNK-mediated transformation by causing G1 arrest (Langenfeld, et al. (1996) supra). This RA effect is associated with down-regulation of cyclin E and cyclin E-associated cdk2 kinase activity (Langenfeld, et al. (1996) supra). Further, RA represses cyclin E expression via activation of a proteosomal degradation pathway (Dragnev, et al. (2004) Clin. Cancer Res. 10:2570-7). These findings suggest proteolysis of cyclin E as an important cancer chemopreventive mechanism since the retinoid-induced G1 cell cycle arrest is expected to permit repair of carcinogenic damage to DNA. The critical role of cyclin E in RA response was confirmed in RA-resistant HBE cells with deregulated cyclin E expression (Dragnev, et al. (2004) supra).
The cyclin E mutant protein (cyclin E-Thr380Ala) stabilizes cyclin E and increases its associated kinase activity (Spruck, et al. (1999) supra). Moreover, overexpression of cyclin E (Thr380Ala) induces chromosome instability at a higher frequency than that observed in cells overexpressing wild-type cyclin E (Spruck, et al. (1999) supra). A cyclin E double mutant (Thr62Ala/Thr380Ala) is more stable than the Thr380Ala mutant (Nakayama, et al. (2000) EMBO J. 19:2069-81). Unlike wild-type cyclin E, which undergoes proteasome degradation, the cyclin E double-mutant protein (Thr62Ala/Thr380Ala) is stabilized after RA-treatment (Dragnev, et al. (2004) supra).